Separation of mixtures of chemicals, biomolecules and cell types is often effected by immunoaffinity chromatography. Packed beds, such as those used in column chromatography, are often used in affinity separation. However, problems such as non-specific trapping or filtration of cells and clogging make the use of a packed bed undesirable for cell separation. In addition, when fine particles are used to increase the mass transfer efficiency of packed beds, a large pressure drop across the bed often results. These problems require significant washing of the packed bed in order to flush contaminants and other cellular debris from the column.
One device that has been developed for reducing the pressure drop across a column of particles is the fluidized bed. A fluidized bed consists of solid particles and a gas or liquid which is passed upwardly through the particle bed with velocity sufficient to ensure that the drag forces of the fluid counterbalance the gravitational forces on the particle and cause random motion of the particles. The bed of particles will become fluidized and expand, resulting in a lower pressure drop across the fluidized bed as compared to the pressure drop across a packed bed of the same height. The fluidization of the bed also provides more surface contact between the particle and the fluid passing through the bed.
One disadvantage associated with fluidized beds is the radial and axial movement of the particles which result in significant intermixing of the particles. An advancement in fluidized bed technology is the magnetically stabilized fluidized bed (MSFB) which involves the use of a magnetic field and magnetizable particles to stabilize the bed. It has been found that, by supplying a magnetic field parallel to the path of fluid flow, the magnetizable particles can be locked in place, thus eliminating the intermixing of the particles.
In general, the MSFB combines some of the best characteristics of the fluidized bed with those of a fixed bed. More particularly, the MSFB provides a low pressure drop, the ability to transport solids through a system and good mass transfer driving force even as the fluid is depleted of its source. See Burns, Structural Studies of a Liquid-Fluidized Magnetically Stabilized Bed, Chem. Eng. Comm, 67:315-330 (1988). All documents cited herein are hereby incorporated by reference.
Because of these advantages, MSFBs have been used to separate various chemical species and proteins, and to filter yeast. For example, for the use of a MSFB to separate proteins see Burns, et al., Application of Magnetically Stabilized Fluidized Beds to Bioseparations, Reactive Polymers, 6:45-50 (1987) (human serum albumin); Lochmuller et al., Affinity Separations in Magnetically Stabilized Fluidized Beds: Synthesis and Performance of Packing Materials, Separation Science and Technology; 22:2111-2125 (1987) (trypsin); and U.S. Pat. No. 5,130,027, issued to Noble, Jul. 14, 1992, (Cytochrome-C). The use of a MSFB to separate various organic and inorganic compounds is discussed in U.S. Pat. No. 5,084,184 issued to Burns on Jan. 28, 1992. Finally, the use of an MSFB as a filter to collect yeast cells was reported by Terranova et al. Continuous Cell Suspension Processing using Magnetically Stabilized Fluidized Beds, Biotechnology and Bioengineering, 37:110-120 (1991). In this; latter reference, the filtration was not based on immunoaffinity but rather on electrostatic interaction between the positively charged nickel particles contained in the MSFB and the negatively charged yeast cells.
However, none of these references discuss the use of the MSFB for the affinity separation of mammalian cell population from a mixture of cell populations. The separation of a particular mammalian cell population from a mixture of cell populations is quite different from the separation of chemical species such as proteins from a solution. Most mammalian ,cells are on the order of 8 to 20 microns (.mu.) in diameter. In contrast, the proteins and other chemical species which have been separated in a MSFB to date are significantly smaller, i.e. on the order of 1000 fold or more. Thus, the probability that the larger mammalian cell with greater fluid drag will bind to the particle and be retained is significantly lower under similar conditions. In addition, another factor unique to the separation of mammalian cells is the need to preserve cell viability.
In contrast to yeast cells, which are relatively insensitive to changes in osmolarity, pH and shear, higher order mammalian cells are much more sensitive to shear forces exerted during purification, pH osmolarity, and the chemical composition of the reagents used. Therefore, both the steps comprising the method and all reagents used must be non-toxic to the cells.
Mammalian hematopoietic (blood) cells provide a diverse range of physiological activities. Blood cells are divided into lymphoid, myeloid and erythroid lineages. The lymphoid lineages, comprising B cells and T cells, provides for the production of antibodies, regulation of the cellular immune system, detection of foreign agents in the blood, detection of cells foreign to the host, and the like. The myeloid lineage, which includes monocytes, granulocytes, megakaryocytes as well as other cells, monitors for the presence of foreign bodies, provides protection against neoplastic cells, scavenges foreign materials, produces platelets, and the like. The erythroid lineage provides the red blood cells, which act as oxygen carriers.
Despite the diversity of the nature, morphology, characteristics and function of the blood cells, it is presently believed that these cells are derived from a single progenitor population, termed "stem cells." Stem cells are capable of self-regeneration and may become lineage committed progenitors which are dedicated to differentiation and expansion into a specific lineage.
A highly purified population of stem cells is necessary for a variety of in vitro experiments and in vivo indications. For instance, a purified population of stem cells will allow for identification of growth factors associated with their self-regeneration. In addition, there may be as yet undiscovered growth factors associated (1) with the early steps of dedication of the stem cell to a particular lineage; (2) the prevention of such dedication; and (3) the negative control of stem cell proliferation.
Stem cells find use: (1) in regenerating the hematopoietic system of a host deficient in stem cells; (2) in a host that is diseased and can be treated by removal of bone marrow, isolation of stem cells and treatment of individuals with drugs or irradiation prior to re-engraftment of stem cells; (3) producing various hematopoietic cells; (4) detecting and evaluating growth factors relevant to stem cell self-regeneration; (5) the development of hematopoietic cell lineages and assaying for factors associated with hematopoietic development; and, is a target for gene therapy to endow blood cells with useful properties.
Highly purified stem cells are essential for hematopoietic engraftment including but not limited to that in cancer patients and transplantation of other organs in association with hematopoietic engraftment. Stem cells are important targets for gene therapy, where the inserted genes promote the health of the individual into whom the stem cells are transplanted. In addition, the ability to isolate the stem cell may serve in the treatment of lymphomas and leukemias, as well as other neoplastic conditions. Thus, there have been world-wide efforts toward isolating the human hematopoietic stem cell in substantially pure or pure form.
Stem cells constitute only a small percentage of the total number of hematopoietic cells. Hematopoietic cells are identifiable by the presence of a variety of cell surface protein "markers." Such markers may be either specific to a particular lineage or progenitor cell or be present on more than one cell type. Currently, it is not known how many of the markers associated with differentiated cells are also present on stem cells. One marker which was previously indicated as present solely on stem cells, CD34, is also found on a significant number of lineage committed progenitors. U.S. Pat. No. 4,714,680 describes a composition comprising human stem cells.
Hematopoietic cells are initially obtained as a mixture of a variety of cell populations ("mixture of cell populations"). The cell population to be purified or enriched for is termed herein the "target" cell population. Separation techniques involve successive purification steps relying on the use of affinity matrices to either retain nontarget cells and allow the target cells to flow through (negative selection) or to retain the target cells and allow the nontarget cells to flow through (positive selection).
Typically, hematopoietic cells are separated using negative selection affinity separations which are performed in a batch mode. For example, in processing a normal bone marrow (BM) harvest, it may be desirable to obtain only those cells exhibiting a specific cell surface antigen such as the CD34 antigen (CD34.sup.+ cells) which includes the stem cell population and a variety of other, more differentiated, cells. Typically, only approximately 0.1 to 5% of the total mononuclear cell population in a blood sample express the CD34 antigen. In contrast, cells expressing the CD15.sup.+ cell surface antigen, i.e. CD15 cells, which are more differentiated than stem cells, comprise approximately 50 to 75% of the total mononuclear cells.
In order to separate the target CD34.sup.+ cells from the mixture of cell populations by negative selection, the cell sample is placed in a vessel containing beads conjugated to an antibody specific to CD15. CD15.sup.+ cells bind to the anti-CD15 antibody and are then removed from solution by removal of the beads. Thus, by depleting the CD15.sup.+ cells, less than half to one quarter of the original cells, including the target CD34.sup.+ cells, remain for additional processing. Negative selection has been essential in separating stem and/or progenitor cells from BM or other sources since the target cells are present in such a low concentration.
In contrast, positive selection refers to a process in which the target cell population is bound to a particle having affinity for the target cell population and the nontarget cell populations do not bind and flow through. The target cell population is then obtained by releasing the cells from the particles and collecting the cells.
Because the target cell population typically comprises only a small fraction of the mixture of cell populations, positive selection would be the preferred process if it could be made efficient enough to selectively remove such a small percentage of cells. As noted earlier, CD34.sup.+ cells comprise only approximately 0.1 to 5% of the total mononuclear cell population. Of these cells stem cells comprise only a small percentage. Considering the time and reagents needed to stain and sort cells, it would be advantageous to directly select CD34.sup.+ cells for subsequent staining and sorting instead successive purification of the mixture of cell populations that typically result from negative selection methods.
Until recently, positive selection of human hematopoietic cells has not been possible. One method which has been developed is the Ceprate LC.RTM. system which uses a packed bed column and an avidin-biotin affinity system to select CD34.sup.+ cells. In this system, the avidin protein is attached to a bead or other solid support. The suspension containing the target CD34.sup.+ cells is mixed with a biotin-conjugated anti-CD34-antibody under conditions which allow the antibody to bind to CD34.sup.+ cells. This suspension is then passed downwardly through the column and, due to the affinity of the biotin to avidin, the CD34.sup.+ cells adhere to the support beads. After the entire suspension has passed through the column, the column is washed to remove any excess suspension or impurities. The support beads with the attached CD34.sup.+ cells are then agitated to physically separate the CD34.sup.+ cells from the beads. Although this method allows for positive selection of CD34.sup.+ cells, it also results in significant non-specific separation and therefore retention of nontarget cells, such as cancerous cells. It is believed that at least a portion of this contamination occurs due to filtration in the packed column.
Accordingly, it is an object of this invention to provide a method for both the positive and negative selection of mammalian hematopoietic cells that results in significantly less non-specific cell separation and filtration.